Cells grown overnight in YNB medium were washed twice in 1× PBS and resuspended to a final concentration of 2.5 × 106 cells/ml in 1× PBS. Ten microliters of cell suspension was spotted onto l -3,4-dihydroxyphenylalanine (l -DOPA)-containing agar or Christensen's urea agar (catalog no. 27048; Sigma). Plates were checked daily for changes in melanization (brown/black colonies on l -DOPA) and urease secretion (pink area surrounding colonies on Christensen's urea).
Christensen s urea agar
Manufactured by Merck Group
Sourced in Germany
Christensen's urea agar is a microbiological culture medium primarily used for the identification and differentiation of urease-producing microorganisms. It is formulated to detect the presence of the enzyme urease, which is produced by certain bacteria as they metabolize urea.
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Lab products found in correlation
Lysine iron agar, by Merck Group (2 mentions)
Triple sugar iron agar, by Merck Group (2 mentions)
Lactose broth, by Merck Group (1 mentions)
Hektoen enteric agar, by Thermo Fisher Scientific (1 mentions)
Tryptic soy agar, by Merck Group (1 mentions)
Xylose lysine deoxycholate agar xld, by Thermo Fisher Scientific (1 mentions)
Sulfide indole motility medium, by Merck Group (1 mentions)
Buffered peptone water (bpw), by Merck Group (1 mentions)
Bismuth sulphite agar, by Merck Group (1 mentions)
Salmonella shigella agar, by Merck Group (1 mentions)
Sulfide indole motility, by Merck Group (1 mentions)
4 protocols using christensen s urea agar
1
Melanization and Urease Activity Assay
2
Growth Characterization of C. gattii Isolates
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To determine the growth rate of C. gattii VGV, cells of all six VGV isolates were inoculated in YEPD broth and incubated at 30°C on a shaker (200 rpm) for 18 h. Cells were washed with sterile phosphate-buffered saline (PBS), and 2 × 105 cells/ml were resuspended in PBS. Three-microliter aliquots of 10-fold serial dilutions were spotted onto YEPD agar and incubated at 30°C and 37°C. For biological confirmation of the species, isolates were inoculated on canavanine glycine bromothymol blue (CGB) agar (80 (link)) for species-specific CGB reactions and on Christensen’s urea agar (Sigma) and norepinephrine agar (81 (link)) for urease and melanin production reactions, respectively, and were incubated at 30°C for 48 h. India ink staining of the cells grown on YEPD broth for 24 h at 30°C was used for microscopic observation of the cell and polysaccharide capsule size. The reference strains used were WM148 or H99 (serotype A, VNI), WM626 (serotype A, VNII), WM179 (serotype B, VGI), WM178, R265 and R272 (serotype B, VGII), WM161 (serotype B, VGIII), and WM779 (serotype C, VGIV) (14 (link)). The mating type of each isolate was determined by PCR using primers specific to STE12α and STE20a (82 (link)).
3
Isolation and Identification of Salmonella spp.
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Isolation of Salmonella spp. was firstly performed by pre-enrichment of samples in lactose broth (Merck, Darmstadt, Germany) at 37 °C for 24 h. For selective enrichment, pre-enriched cultures were transferred into Selenite Cystine (SC) broth (Merck, Darmstadt, Germany) and Tetrathionate Brilliant Green bile (TBG) broth (Merck, Darmstadt, Germany), and incubated at 35 °C for 24 h. Then, these cultures were streaked onto Bismuth Sulphite agar (BSA) (Oxoid, Basingstoke, UK), Xylose Lysine Deoxycholate Agar (XLD) (Oxoid, Basingstoke, UK), and Hektoen Enteric agar (HEA) (Oxoid, Basingstoke, UK) as selective media and incubated at 35 °C for 48 h. Typical colonies were cultured on the slants of Tryptic soy agar (TSA) (Merck, Darmstadt, Germany) and subjected to biochemical tests using Lysine Iron agar (LIA) (Merck, Darmstadt, Germany), Triple Sugar Iron (TSI) agar (Merck, Darmstadt, Germany), Sulfide-Indole-Motility (SIM) medium (Merck, Darmstadt, Germany), and Christensen’s Urea agar (Merck, Darmstadt, Germany) (Table S1 ) [89 ].
4
Isolation and Identification of Salmonella
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The eggs were transferred aseptically to beakers containing 225 ml of Buffered Peptone Water (BPW, Merck, Germany). They were then incubated at 37 °C for 24 h followed by transferring of 1 ml to the selenite–cystine broth (SC, Merck, Germany) and 0.1 ml to the Rappaport–Vasiliadis medium (RV, Merck, Germany) with incubation for 24 h at 37 °C (SC) and 41.5 °C (RV) (23 (link)). From the broths one loopful was subcultured on Brilliant Green and Phenol Red agar (BGA, Merck, Germany) and Bismuth Sulphite Agar (BSA, Merck, Germany). The media were inoculated at 37 °C for 24 h (BGA) or 48 h (BSA). Then, suspected colonies were transferred onto Salmonella-Shigella agar (Merck, Germany) plates, and incubated at 37 °C for 24 h. The plates were observed for typical Salmonella-like colonies, randomly, two colonies from each plate were picked, purified and subjected to primary biochemical screening tests, which involved reactions on Triple Sugar Iron agar (Merck, Germany), Lysine Iron agar (Merck, Germany), motility and Indole and H2S production in Sulfide-Indole-Motility (SIM, Merck, Germany) and urea splitting ability in Christensen’s Urea agar (Merck, Germany). Slide agglutination test were performed using polyvalent H serum and the group sera (Difco, USA).
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